Method of promoting mold growth



June 1944- L. M. CHRISTENSEN 68 METHOD OF PROMOTING MOLD GROWTH Filed Oct. '7, 1940 4 Sheets-Sheet 1 I DILUTE ACID SUCH AS HYDRO- :74/ CHLORIC 0 LACTIC ACID ADDING PHOSPHATE SUCH As PHOS- FIBROUS MATERIAL SUCH AS PHOR|C ACID o SQLUB'LE PHOS- WHEAT BRAN PHATES IF AVAILABLE PHOSPHATES IN BRAN ARE INADEQUATE IF BRAN IS DEFICIENT IN THESE COMPOUNDS E MIXING THE BRAN WITH TREATED ACID STERILIZING- i COOLING INOCULATING wITI-I MOLD SPORES I mcuBATms INNOCULATED MATERIAL IN Two STAGES,

WITH AND WITHOUT AERATION BREAKING THE MATS FORMED IN INCUBATION STAGES FURTHER AERATms" I PRODUCT OF HIGH o'IAsTATIc PROPERTIES GRINDING FOR USE IN DRYING FOR STORAGE- FERMENTATION PROCESSES Qvwe/wtoo LEO M. CHRISTENSEN June 1944' L. M. CHRISTENSEN 2,352,168

METHOD OF PROMOTING MOLD GROWTH Filed 001:. 7, 1940 4 Sheets-Sheet 2 WHEAT BRAN MOISTENING'WITH EQUAL 6. Z PARTS BY WEIGHT OF WATER ADDING SOLUBLE PHOSPHATE IF BRAN IS DEFICIENT IN AVAILABLE PHOSPHATES STERILIZING ADDING PREPARED BRAN MOLD OR OTHER SUITABLE DIASTATIC MATERIAL HEATING AT CONTROLLED TEMPERATURE TO CONVERT I STARCH PRESENT TO FERMENTABLE CARBOHYDRATES COOLING INOCULATING WITH CULTURE OF LACTOBAC/LLUS CASE] OR OTHER LACTIC ACID PRODUCING BACTERIA RESTERILIZATION IF BUTYRIC ACID CONTAMINATION IS PRESENT 4 I INCUBATING I INOCULATING WITH SPORES OF MOLD I CULTURE SUCH AS ASPERG/LLUS ORYZAE I BREAKING MATS FORMED IN INCUBATION FURTHER AERATION awe/WM I PRODUCT OF HIGH LEO M. CHRISTENSEN DIASTATIC PROPERTIES June 27, 1944. c s s 2,352,168

' METHOD OF PROMOTING MOLD GROWTH Filed OGIZ. 7, 1940 4 Sheets-Sheet 3 FIBROUS MATERIAL OTHER THAN DILUTE ACID SUCH AS HYDRO- WHEAT BRAN SUCH AS EX- CHLQRIC OR LACTIC ACID TRACTED SOYBEAN MEAL, CORN BRAN, SAWDUST, ETC. OR

- BARLEY MALT I ADDING METALLIC SALTS SUCH Y AS SALTS OF ZINC, IRON OR COPPER ADDING FERMENTABLE CARBO- HYDRATES SUCH AS STARCH OR SUGAR IF MATERIAL IS -LOW IN I CARBOHYDRATE CONTENT ADDING SOLUBLE PHOSPHATE INOCULATING WITH MOLD SPORES INCUBATING IN TWO STAGES-WITHOUT AERATION FOLLOWED BY AERATION BREAKING UP MATS FORMED IN INCUBATION FURTHER AERATION PRODUCT OF HIGH DIASTATIC PROPERTIES gvvuq/wbo'n LEO M. CHRISTENSEN A abto'wan o J1me 1944- L. M. CHRISTENSEN I 2,352,168

METHOD OF PROMOTING MOLD GROWTH Filed Oct. 1940 4 Sheets-Sheet 4 SOYBEANS 0R BARLEY MALT TREATING WITH A BACTERIA WHICH WILL PRO- DUCE A DIASTATIC ENZYME SYSTEM OF HIGH ALPHA AMYLASE YCONTENT SUCH AS ONE OF THE BAC/LLUS 'MESE/VTER/CUS GROUP .INOCULATION WITH MOLD SPORES INCUBATING IN TWO STAGES-WITHOUT AERATION FOLLOWED BY AERATION BREAKING UP MATS FORMED IN INCUBATION FURTHER AERATION PRODUCT OF HIGH DIASTATIC PROPERTIES gwumm LEO M. CHRISTENSEN MO -mango 2 MW, @140? @mm Patented June 27, 1944 UNITED STATES PATENT OFFICE METHOD OF PROMOTING MOLD GROWTH Leo M. Christensen, Moscow, Idaho, assignor to National Agrol Company, Inc., New York, N. Y., a corporation of Delaware Application October 7,19,40, Serial No. 360,206

7 Claims.

This invention generally relates to a method of facilitating the growth of molds on solid substrates of fibrous character, and more especially to a method of supplying and rendering available to the mycelium the nutrients required to promote mold growth.

Hitherto, wheat bran has been generally found to be the most satisfactory substrate for the production of diastatic preparations. I have found thatthis is partly due to the fact that this bran ordinarily contains inorganic nutrients, especially salts of the heavy metals which are needed for the best growth of mold, such as Aspergillusoryzae. Wheat bran is also relatively rich in phosphate containing constituents which are required for the proper growth of molds of this type. Moreover, the usual carbohydrate content of wheat bran is approximately that needed for best mold growth. V

However, wheat brans from various sources vary considerably in their value as a substrate for the production of diastatic preparations, and the mold brans made from these various brans vary as much as 50% in their diastatic power. In this connection, I have found that this is occasioned almost entirely by the variation in the contents of the various wheat brans of the required mold nutrients, particularly heavy metals. Moreover, much of the phosphate present in wheat bran is in organic combination and as such is not water soluble and therefore is'not readily available.

One of the objects of this invention is to provide a method by which the substrates of fibrous material other than wheat bran may be used to produce diastatic preparations.

Another object of this invention is to provide a method of making available the insoluble phosphates contained in fibrous materials such as wheat bran.

A further object of this invention is to supply to fibrous material in proper proportions the phosphates and metal salts necessary to promote mold growth.

With these and other objects in view, this invention embraces broadly the idea of preparing fibrous material, such as wheat bran, to increase its efilciency for the production of diastatic preparations. The method varies with the type of fibrous material which is selected. For example, if a fibrous material, such as wheat bran which is rich in organic phosphates, is selected, it is only necessary to render these phosphates available to the mold mycelium. In other types 01' fibrous material, such as extracted soybean,

per c. c. may be used.

sawdust and the like, or in poor grades of wheat bran it is necessary to add available phosphates. Moreover, many grades of wheat bran and most other usable materials contain insuflicient quantitles of phosphate and metallic salts and these insufllciencies must be corrected.

In the drawings:

Figure 1 discloses one method'of practicing my invention.

Figure -2 discloses a second method of practicing my invention.

Figure 3 discloses a third method of practicing.

my invention.

Figure 4 discloses a fourth method of practicing my invention.

In Figure 1 is disclosed a method of treating 'wheat bran to produce a material having diastatic properties. In this form of the invention, a dilute acid is added to make the phosphates contained in the wheat bran water soluble. Preferably, a solution of hydrochloric acid is used although lactic acid may be employed if de sired. Sulphuric acid has not been found suitable for this use.

If hydrochloric acid is selected, a dilute solution of from 0.05 to 0.25 normal is added to the bran and the mixture is subjected to heat for a period of from thirty to sixty minutes at temperatures ranging from 100 to C. The dilute acid is added in the proportions of one part acid to one part of wheat bran.

While'this treatment renders the phosphates available and therefore effects a marked improvement in the mold growth obtained, the amount of soluble phosphate thus produced is still inadequate in some brans. For this reason, if necessary, phosphoric acid or equivalent amounts 'of water soluble phosphates may be added to the dilute acid to correct this deficieney. The optimum phosphoric acid addition is approximately 0.25 gram per 100 c. c. of the dilute acid but, if desired, as much as 1.0 gram The beneficial effects of the addition of phosphoric acid or phosphates is evidenced by one experiment in which the amyloclastic value of a mold bran made from. a bran deficient-in phosphates was increased from fifteen to twenty by the addition of 0.25 gram of phosphoric acid per 100 c. c. of dilute hydrochloric acid to moisten the bran. That is, the amount of starch saccharified by one gram of the dry mold bran was increased one-third by the addition of this amount of phosphoric acid.

In practicing the process, it is also desirable to add metallic salts if the bran is deficient in these nutritive compounds. For example, it may be found necessary to add zinc, copper and iron salts to the dilute acid used to moisten the bran. I have found that the amount of added salts required varies somewhat depending on the grade of bran used but, except in a few instances, the addition of 0.000625 gram of crystalline zinc sulphate, 0.00080 gram of crystalline copper sulphate, and 0.000625 gram of crystalline ferrous sulphate per liter of the dilute acid used to moisten the bran is optimum. That is to say, that these additions are adequate for most deficient brans and are not excessive in the case of brans of higher heavy metal content.

The beneficial effect of these salt additions is aeration, until a temperature of C. is reached.

The spores of the mold are added and the mate- -rial is subjected to incubation.

Preferably, this incubation consists of a twelve hour period without aeration and then a twelve hour period in which the temperature produced by the oxidative processes is controlled by aeration and is maintained at less than 50 C. and preferably between 40 and C.

After twenty-four hours, the mycelium is fully rown and forms the material into a solid mat. This mat is broken into pieces which are roughly an inch in diameter and the material is again aerated for approximately twenty-four hours.

After this treatment, the product may be dried and stored or it may be ground and used in a fermentation process to convert starchy materials to carbohydrates. If desired, water may be added and the product made into a slurry before it is added to the starchy materials which are to be treated.

Although the above process is a highly satisfactory means of producing a product having high diastatic properties, I have found that the use of lactic acid is just as satisfactory and has the advantage that it can be made from the carbohydrates present in the bran which otherwise are oxidized into carbon dioxide and water by the mold. A flow sheet of this process is disclosed in Figure 2 of the drawings.

In practicing this process, the bran is first moistened with an equal weight of water and is sterilized at a temperature of from 100 to 110 C. for thirty tosixty minutes in the manner previously described. If the material contains an insufficient quantity of phosphates and heavy metal salts, these are added to the bran before sterilization, the amounts used being substantially the same as those disclosed in the first process.

The bran is then cooled to approximately 55 C. and a previously prepared mold bran or other suitable diastatic material is added. Usually the amount required consists of from 1 to 5% of the dry weight of the bran. After the mold bran has been added, the temperature of 55 C. of the mixture is maintained by heating if necessary for from one hundred and twenty to one hundred and fifty minutes in order to convert substantial amounts of the starch present in the bran into fermentable carbohydrates.

The bran is then cooled to approximately 30" C. and is inoculated with a culture of Lactobacillus casei or other suitable lactic acid producing bacteria, and, if the above'mentioned bacteria is used, the mixture is incubated at 30 C. for about twenty-four hours. Obviously, other lactic acid cultures may require a slightly different temperature and time of incubation.

At the end of twenty-four hours of incubation or as soon as the acidity of the bran has reached a pH of 4.0, it is inoculated with spores of Aspergillus oryzae or other suitable mold culture. The mixture is then subjected to the incubation and subsequent steps mentioned in connection with the process shown in Figure 1.

In this connection it should be noted that ordinarily it is not necessary to re-sterilize the bran after the lactic fermentation and prior to the inoculation with the spores of the mold. In fact, there are growth stimulants formed by the lactic bacteria which have a beneficial effect upon the mold as evidenced by the rapid growth of mold mycelium, especially during the first twelve or twenty-four hoursof incubation. However, if there should be contamination of the lactic fermentation with butyric acid bacteria, it is necessary to resterilize before incubation. Provision, therefore, should be made for resterilization in the occasional case of such contamination.

The process disclosed in Figure 3 of the drawings relates to the growth of mold on fibrous maeither in Figure 1 or 2 except that the available phosphates and the salts of heavy metals must be added.

In Figure 4 is also disclosed a method of producing a good mold growth on barley malt or a soybean material. In this process sterilization prior to inoculation with the mold spores is undesirable, and by maintaining a pH of from 4.0 to 4.2 the growth of bacteria harmful to the process is suiilciently inhibited to avoid any difiiculty.

In this manner, a dias'tatic preparation incorporating the diastatie system of barley malt and that of mold included'in one product may be readily prepared. Soybeans, either unpressed or after the lecithin and oil have been removed, may

also be treated in this manner to produce mixed diastatic systems.

2 except that the sterilization is avoided as has previously been described.

While for purposes of illustration I have disclosed four processes for developing a product having improved diastatic properties, it is obvious that various changes can be made in the order of the procedure in the processes and various changes made in the temperature and periods of treatment without departing from the scope of the invention.

I claim:

1. A method of facilitating the growth of a diastatic mold on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid to the said fibrous material, sterilizing and then cooling the result ing mixture, adding spores of the said mold, incubating the inoculated material without aeration, and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidation.

2. A method of facilitating the growth of a diastatic mold on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid to the said fibrous material, sterilizing and then cooling the resulting mixture, adding spores of the said mold, in-

cubating the inoculated material for a period of twelve hours without aeration, and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidation.

3. A method of facilitating the growth of a diastatic mold on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid, available phosphates and salts of heavy metals to the said fibrous material, said salts constituting less than 1% by weight of the total mixture, sterilizing and then cooling the said mixture, adding spores of the said mold, incubating the inoculated material without aeration and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidation.

- 4. A method of facilitating the growth of a diastatic mold on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid, available phosphates and salts of heavy metals to the said fibrous material,.said salts constituting less than 1% by weight of the total mixture, sterilizing and then 3 cooling the said mixture, adding spores of the said mold, incubating the inoculated material for twelve hours without aeration and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidaweight of the total mixture, sterilizing and then cooling the said mixture, adding spores of the said mold, incubating the inoculated material for.

twelve hours without aeration and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidation so as to maintain a temperature within a range of from 40 to 50 centigrade.

6. A method of facilitating the growth of mold of the species Aspergillus oryzae on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid, available phosphates and salts of heavy metals to the said fibrous material, said salts constituting less than 1% by weight of the total mixture, sterilizing and then cooling the said mixture, adding spores of the said mold, incubating the inoculated material for twelve hours without aeration and then fermenting the inoculated material for an equal period of time with aeration to remove the heat of oxidation so as. to maintaina temperature within a range of from 40 to 50 centigrade.

'7. A method of facilitating the growth of a diastatic mold on solid substrates of carbohydrates of fibrous character comprising adding a dilute solution of hydrochloric acid, available phosphates and salts of heavy metals to the said fibrous material, said salts constituting less than 1% by weight of the total mixture, introducing a fermentable carbohydrate in sufficient amounts to constitute at least 10% of the total dry weight, 

